The active HIV-1 protease is a homodimer made up of monomers containing 99 amino acid residues. All drugs directed against the protease have targeted the substrate-binding site of the dimer. Although at least six drugs have been developed that binding tightly to the active site, one unfortunate consequence of targeting a single drug binding site on the protease has been the emergence of viral strains which carry multi-drug resistant mutations on their protease constructs. For this reason, there is considerable interest in identifying other protease sites that are suitable drug targets. Although the protease monomer is completely inactive, we have demonstrated that it is folded and therefore contains numerous potential alternative drug-binding sites, making it an attractive anti-HIV target. In order to overcome problems with monomer aggregation, we have designed proteins in which the N- and C-terminal regions are linked by intra-monomer disulfide bonds. In particular, cysteine residues were introduced at positions 2 and at either 97 or 98 of the protease amino acid sequence and using NMR have shown that the Q2C/L97C monomer construct exhibits a fold similar to that observed for the monomer subunit in the active dimer. It is anticipated that monomeric proteases of this kind will aid in the discovery of novel inhibitors, that bind to the monomer at the dimerization interface, rather than at the active site. Such inhibitors could circumvent the problem of multidrug-resistance invariably observed with current HIV-1 protease drugs all of which bind at the active site. We have extend thses studies of the protease monomer by using NMR to determine the first solution structure of a protease monomer, the HIV-1 protease spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease monomer is essentially identical to that of the individual subunit of the active protease dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric exhibiting a tertiary fold that is quite similar to the PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N-terminus as compared to the mature protease, which is an active stable dimer under identical conditions. An addition of even 2 amino acids to the N-terminus of the mature protease significantly increases its dissociation into monomer. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor. We have advanced NMR methodology for studying slow protein motions (on the millesecond to microsecond timescale) in solution by developing pulses sequences that measure the relaxation dispersion of amide proton and backbone carbonyl spins in proteins. These measurements together with measurements of NMR lineshapes, carried out at temperatures ranging from 2 to 28 ?C, and measurements of hydrogen-deuterium exchange are being analyzed with the goal of developing a detailed model of the conformational fluctuations that occur at the dimer interface of the protease, both when it is free and when it is bound to a potent inhibitor.